Broad goals are to define the mechanisms of blood clotting by Staphylococcus aureus staphylocoagulase (SC) and von Willebrand factor binding-protein (vWbp), and their roles in acute infective endocarditis (AIE). Endothelial damage on heart valves activates coagulation and concurrent S. aureus bacteremia allows bacteria to bind to the injury site. SC/vWbp activate host prothrombin (ProT) non-proteolytically by inserting their N-termini into a cleft in ProT. Active SC/vWbp.ProT* complexes cleave fibrinogen (Fbg) to fibrin (Fbn), aiding growth of platelet-Fbn-bacteria vegetations. S. aureus AIE is 25-47% fatal despite antibiotic therapy. Due to rapid spread of hypervirulent, multidrug-resistant strains, S. aureus wil likely become resistant to all antibiotics and a huge threat to public health. Drug companies are reluctant to develop new antibiotics due to low profitability, exacerbating the threat. Aim 1 will define the specificity of ProT for insertion of the SC N-terminus. The 3 N-terminal residues are hypothesized to dictate affinity and activity of SC.ProT*. Virtual screening of 3- residue combinations will select the highest affinity for evaluation in 3 contexts: SC, vWbp, and meizothrombin. Aim 1 answers the question: Does the conservation of Ile-Val and Val- Val N-termini in serine proteinases reflect the strict specificity of the binding cleft, or are there resdue combinations with higher affinity? SC results show that the ProT cleft accepts diverse residues, some producing higher activity than wild-type. Likely outcomes are that activity and affinity are context-dependent and the bases for the differences will be defined. An anti-SC(1-10) mAb inhibits ProT activation with nM affinity, increases survival in a sepsis model, and supports the potential to develop mAb therapeutics for AIE. Aim 2 will define the binding and activation pathway of ProT by SC and Fbg binding to SC.ProT*. Rapid-reaction kinetics will access millisecond-second time-scales on which these events occur. Outcomes will identify the activation step for ProT and the step of Fbg substrate engagement. Aim 3 will decipher the roles of the disordered C-terminal pseudo-repeat and seven, 27-residue SC repeats in binding fibrin(ogen) and fragment D. Binding sites on the repeat sequences and fragment D will be mapped by crosslinking. Fbg binding and cleavage kinetics will test if the repeats assist Fbg binding through a (SC.ProT*)2.Fbg complex. Anti-repeat mAbs will be evaluated in vitro and in vivo. Aim 4 tests the idea that SfbX and ZAAP4 activate ProT by N- terminal insertion and will discover the substrates and adhesion proteins of the SfbX/ZAAP4.(pro)thrombin* complexes, providing insight into their roles in pathogenesis.